Mass spectrometry consists in "weighing" individual molecules by transforming them intact into ions in vacuo and then measuring the response of their trajectories to various combinations of electric and/or magnetic fields. Attempts to extend the application of mass spectrometric methods to the analysis of very large polar organic and bio-organic molecules have long been frustrated by the difficulties of transforming such molecules into ions. The analytical advantages of mass spectrometry for such parameters as detection sensitivity, accuracy of mass measurement and abundance determinations cannot be realized if the prerequisite ions cannot be formed. Large polar molecules generally cannot be vaporized, even in vacuo, without extensive, even catastophic, decomposition. Consequently, one cannot apply the classical methods of ionization based on gas phase encounters of the molecule to be ionized with electrons as in Electron Ionization (EI), photons as in Photo Ionization (PI), other ions as in Chemical Ionization (CI), or excited atoms or molecules as in Auger Ionization (AI). Such encounters can form ions from a neutral molecule by a variety of mechanisms including removal or attachment of an electron and removal or attachment of a positively charged entity, typically a proton.
In recent years a number of so-called "soft" ionization methods have been developed which with varying degrees of success have been able to produce intact ions from molecular species of ever increasing size. One class of such methods is based on very rapid deposition of energy on a surface over which the species to be analyzed (analyte) has been dispersed. The idea is, as first suggested by Beuhler et al, Journal of American Chemical Society, 96, 3990 (1974), that if the heat required is applied rapidly enough, vaporization may occur before decomposition has a change to take place. The rapid heating methods now in use include Plasma Desorption (PD), in which disintegration of a radioactive isotope, usually Californium-252, produces a small blob of plasma on the surface from which a few intact ions of analyte emerge; Secondary Ionization Mass Spectrometry (SIMS), hereafter referred to as Fast Ion Bombardment (FIB), in which the analyte-containing surface is bombarded by ions, e.g., Cs+, accelerated to energies in the tens of kilovolts; Fast Atom Bombardment (FAB) in which the accelerated ions are neutralized by charge exchange before they strike the surface; Laser Desorption (LD) in which photons comprise the vehicle for depositing energy on the surface. These methods have been able to produce intact ions from remarkably large analyte species even though, except for LD, they are high irreversible and characterized by brute force. To date, intact ions have been produced from bio-organic compounds with molecular weights on the order of 210,000 with LD (M. Karas and F. Hillenkamp, paper presented at 11th International Mass Spectrometry Conference, Bordeaux, France 1988; cf. Analytical Chemistry (1988) 60, 2299), 24,000 with FAB (or FIB) (M. Barber and B. N. Green, Rapid Communications in. Mass Spectrometry. (1987) 1, 80) and 45,000 with PD (G. Jonsson, P. Hakansson, A. Hedin, D. Fenyo, B. Sundqvist, H. Bennich and P. Roepstorff, Rapid Commun. Mass Spectrom. in press). The ion currents in these methods have been very small and except for LD decrease rapidly with increasing molecular weight. When the ions get very large their detection with multipliers requires post-acceleration voltages that are often awkwardly high. Except possibly with LD, the ions produced often have high levels of internal excitation which can result in substantial peak broadening due to predissociation.
Quite different in practice and principle from these "violent" ionization methods are techniques that use very strong electrostatic fields to extract ions from a substrate. In so called Field Desorption (FD) ionization the analyte molecules are applied to a fine wire on whose surface is disposed an array of sharp pointed needles or "whiskers." When the wire is placed in a vacuum system and a high voltage is applied while it is carefully heated, the analyte molecules will desorb as ions from the tips of the needles where the effective field strength is very high. Even though it can transform very involatile analytes into ions in vacuo FD has not become widely used, in part because sample preparation is tedious, in part because of difficulties in adjusting the wire to just the right temperature and voltage, and in part because the desorbed ions have such high energies that relatively expensive magnetic sector analyzers must be used for mass determination. In so-called Electrohydrodynamic Ionization (EH) analyte is dissolved in a non-volatile liquid (e.g. glycerol) and injected into an evacuated chamber through a small capillary tube maintained at high voltage. The solvent liquid must have a low vapor pressure so that it won't "freeze-dry" from rapid evaporation into vacuum. Solute ions, along with molecules and clusters of solvent, are desorbed from the emerging liquid by the high field at its surface and can be mass analyzed. EH has not been widely practiced, in part because few liquids that have low vapor pressure are good solvents for large polar bio-organic compounds, in part because the desorbed ions are usually solvated with one or more molecules of the solvent, and in part because they often have a wide distribution of energies. Moreover, as in the case of FD, the production ions have high energies and require magnetic sector analyzers.
In the past few years there has emerged a new family of ionization techniques that also make use of high electric fields to desorb ions. These techniques differ from FD and EH in that desorption is from small charged droplets of solution into an ambient bath gas instead of into vacuum. The required high fields at the droplet surface result from the increasing charge density and decreasing radius of curvature of the droplet surface as the solvent evaporates. A portion of the bath gas containing the desorbed ions is then admitted through a small orifice into a vacuum system containing an appropriate mass analyzer. The bath gas acts as a very effective moderator, i.e. it maintains both internal and translational energies of the ions at levels corresponding to the bath gas temperature which is rarely high enough to cause thermal decomposition of even labile bio-organic compounds. In Thermospray (TS) ionization which was developed by Vestal and his colleagues the sample solution is passed through a heated tube whose walls are hot enough to vaporize most of the solvent. (J. Amer. Chem. Soc. (1980) 102, 5931). The consequent rapid expansion of solvent vapor produces acceleration and shear forces that atomize the remaining liquid. Thus there emerges from the end of the tube a supersonic jet of superheated solvent vapor in which the remaining sample solution that was atomized is dispersed as small droplets, equal numbers of which are positively and negatively charged. The charging is a result of statistical fluctuations in the distribution of cations and anions as the liquid is nebulized. In a somewhat equivalent technique, called Atmospheric Pressure Ion Evaporation (APIE) by its originators, J. V. Iribarne and B. A. Thomson, droplets are produced by intersecting a flow of sample solution with a high speed jet of air. (J. Chem. Phys. (1976) 64, 2287 and ibid. (1979) 71, 4451). In this discussion APIE will be referred to by the more convenient term Aerospray (AS) to indicate that it is based on pneumatic nebulization of the sample liquid. As in TS the charging is due to statistical fluctuations in the distribution of cations and anions among the droplets during atomization of the liquid. It was found that an induction electrode, at a potential of 3 kilovolts and placed near the atomization region, greatly increased the total ion current. Moreover, all the resulting droplets and desorbed ions had the same sign, positive or negative, depending upon the electrode polarity.
The invention described in this application stems from and relates to so called Electrospray (ES) ionization which can be considered a sort of mirror image of TS and AS in that instead of producing charging by atomization it produces atomization by charging. In ES the liquid sample is introduced through a small bore tube maintained at several kilovolts with respect to the surrounding walls of a chamber containing bath gas, usually but not necessarily at or near atmospheric pressure. The electrostatic field at the tip of the tube charges the surface of the emerging liquid. The resulting coulomb forces overcome the liquid's surface tension and disperse it into a fine spray of charged droplets. Thus, the nebulization is by electrostatic forces that provide a much higher charge/mass ratio for the resulting droplets that can be achieved in TS and AS. If the field at the tip of the tube is too high, or the pressure of the ambient bath gas is too low, a corona discharge will occur at the tip of the tube and substantially decrease the effectiveness of the nebulization. This ES ionization technique is fully described in U.S. Pat. Nos. 4,531,056 and 4,542,293 which were granted in 1985.